Artificial antigen of aflatoxin biosynthetic precursor sterigmatocystin and method for preparing same

ABSTRACT

An artificial antigen of aflatoxin biosynthetic precursor terigmatocystin (ST) and a method for preparing same. Firstly, hydroxyacetic acid is reacted with the double bound of the difuran ring in the aflatoxin biosynthetic precursor ST, yielding an aflatoxin biosynthetic precursor ST hapten with an active carboxymethoxy group. Secondly, a carboxyl group on the ST hapten is attached to an amino group on a carrier protein. At last, the artificial antigen of aflatoxin biosynthetic precursor ST is obtained by dialysis and lyophilize.

CROSS-REFERENCE TO RELATED APPLICATION

This non-provisional application claims priority under 35 U.S.C. §119(a)on Patent Application No. 201410115645.X filed in P.R. China on Mar. 26,2014, the entire contents of which are hereby incorporated by reference.

Some references, if any, which may include patents, patent applicationsand various publications, may be cited and discussed in the descriptionof this invention. The citation and/or discussion of such references, ifany, is provided merely to clarify the description of the presentinvention and is not an admission that any such reference is “prior art”to the invention described herein. All references listed, cited and/ordiscussed in this specification are incorporated herein by reference intheir entireties and to the same extent as if each reference wasindividually incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to an artificial antigen of aflatoxinbiosynthetic precursor ST and a method for preparing same.

BACKGROUND OF THE INVENTION

A fungal toxin is a secondary metabolite secreted by a toxin-producingfungus and a natural toxic compound capable of causing various injurieson the human being and the livestock. Among fungal toxins ever found,aflatoxin (hereinafter referred to as “AFT”) is the most toxic fungaltoxin, and its toxicity, carcinogenicity and contamination frequency allrank the first among biological toxins. Sterigmatocystin, i.e., ST, is aprecursor for aflatoxin synthesis and produced mainly by the fungi suchas Aspergillus versicolor, Aspergillus flavus, Aspergillus nidulans, andAspergillus rugulosus, etc. ST may contaminate most of grains and foragegrasses, and in particular severely contaminate wheat, maize, peanut,forage grasses. The basic structure of aflatoxin biosynthetic precursorST consists of a difuran ring in connection with xanthone. The precursorhas a structure similar to that of aflatoxin, and has toxicity that issecond only to that of aflatoxin. Toxicity of ST includeshepatotoxicity, nephrotoxicity, cytogenetic toxicity and potentcarcinogenicity. It enters into the human food chain after contaminatingfoods and feeds, and poses a threat to the health and safety of human.The hazard degree has a positive correlation with the intake ofaflatoxin biosynthetic precursor ST. Since China is an area where thereis more severe contamination with aflatoxin biosynthetic precursor ST,one of the key points to fortify the food safety is to improve detectionof aflatoxin biosynthetic precursor ST in food products and feeds.Accordingly, it is necessary to determine the content of ST in cerealsand finished products thereof suspected to be contaminated with theaflatoxin biosynthetic precursor ST.

Currently, methods for detecting aflatoxin biosynthetic precursor STmainly include thin-layer chromatography (TLC) and liquidchromatography. TLC is easy to operate, and does not require complex andprecise instrumentation, but has low sensitivity and of low accuracy.Using TLC, a lower limit of detection of aflatoxin biosyntheticprecursor ST in rice, maize, and wheat samples is 25 μg/kg and that insoybean and peanut samples is 50 μg/kg. In the recent years,high-performance liquid chromatography (HPLC) has been used widely inthe detection of fungal toxins and has also been reported for detectingthe aflatoxin biosynthetic precursor ST. However, the application of theHPLC in detection at the basic level has been limited by tediouspre-processing, expensive instrumentation, requirements for stringentoperation environment, and professional operators, etc. Therefore, thereis a pressing need in the detection field in China to study and developnovel techniques for rapid detection of aflatoxin biosynthetic precursorST, which is of importance on guaranteeing the safety in foodconsumption in China.

Immunological analysis techniques have been increasingly the focus ofrapid detection techniques for pollutants such as aflatoxin, due toadvantages such as high sensitivity, short detection time, and easy tooperate, etc. However, there are few reports on rapid detectiontechniques for aflatoxin biosynthetic precursor ST. Antigens andantibodies are the core reagents and the technological sources inimmunological analysis techniques. The aflatoxin biosynthetic precursorST has a molecular weight of 324, belongs to small molecule compounds(<1000) and is incapable of directly stimulating an animal to produceantibodies. Only after covalently coupled to a carrier protein such asbovine serum protein (BSA), egg white albumin (OVA), and polylysine,etc., would aflatoxin be converted into a complete antigen with bothreactogenicity and immunogenicity which can stimulate an animal toproduce antibodies. Currently, antibodies obtained by immunization withan artificial antigen of aflatoxin biosynthetic precursor ST (thesynthesis method is mainly sodium borohydride reduction) have beenreported to be low in sensitivity and poor in specificity.

SUMMARY OF THE INVENTION

In one aspect, the present invention relates to an artificial antigen ofaflatoxin biosynthetic precursor ST and a method for preparing theartificial antigen. Antibodies with high sensitivity and strongspecificity can be produced against aflatoxin biosynthetic precursor ST.The preparation process of the artificial antigen is simple andpracticable, is easy in scale up production, and is easy in widenapplications.

In one embodiment, an artificial antigen of aflatoxin biosyntheticprecursor ST is obtained by coupling the aflatoxin biosyntheticprecursor ST to a carrier protein. The coupling process includes:opening a double bound of the difuran ring of the aflatoxin biosyntheticprecursor ST, and connecting an active carboxymethoxy group, thenconnecting the carboxyl on the active carboxymethoxy group to the aminogroup on the carrier protein.

In one embodiment, a method for preparing the artificial antigen ofaflatoxin biosynthetic precursor ST includes: firstly, reactinghydroxyacetic acid with the double bound of the difuran ring in theaflatoxin biosynthetic precursor ST, to obtain an aflatoxin biosyntheticprecursor ST hapten with an active carboxymethoxy group; then via anactive ester procedure, attaching the carboxyl group on the aflatoxinbiosynthetic precursor ST hapten to an amino group on the carrierprotein at room temperature; and at last, dialyzing and lyophilizing toobtain the artificial antigen of aflatoxin biosynthetic precursor ST.

In one embodiment, the reaction process of the hydroxyacetic acid withthe double bound of the difuran ring of the aflatoxin biosyntheticprecursor ST is as follows: firstly, weighting 0.1-1 g of hydroxyaceticacid and dissolving it in 0.4-4 mL of trifluoroacetic acid; secondly,weighing 1-10 mg of the aflatoxin biosynthetic precursor ST anddissolving it in 0.4-4 mL of acetonitrile, to obtain a solution ofaflatoxin biosynthetic precursor ST in acetonitrile; then, drawing thesolution of aflatoxin biosynthetic precursor ST in acetonitrile with asyringe and pipetting the drawn solution gently into the mixture ofhydroxyacetic acid/trifluoroacetic acid, where the reaction is run atroom temperature for 4-6 h with stirring; after completion of thereaction, evaporating rotationally the solvent to yield a lightyellowish green oily substance, which is the aflatoxin biosyntheticprecursor ST hapten.

In one embodiment, the carboxyl group on the aflatoxin biosyntheticprecursor ST hapten is attached to the amino group of the carrierprotein as follows: weighting 1-10 mg of aflatoxin biosyntheticprecursor ST hapten and 4-30 mg of N-hydroxylsuccinimide and placingthem into a reaction flask and reacting for 1-2 h; weighing 7-45 mg ofcarbodiimide and dissolving it in 0.2-1.2 mL of 1,4-dioxohextane, toobtain a solution of carbodiimide in 1,4-dioxohextane, then adding thesolution dropwise into the reaction flask and reacting at roomtemperature for 4-5 h until a white precipitate is generated in thereaction flask, after completion of the reaction, allowing the reactionmixture to stand at room temperature overnight, and on the next day,centrifuging to obtain the supernatant; adding the obtained supernatantdropwise into a phosphate buffered saline (PBS) solution having 4-30 mgof the carrier protein dissolved therein, after adding the supernatant,running the reaction for 4-5 h, to obtain an complete antigen ofaflatoxin biosynthetic precursor ST in aqueous phase.

In one embodiment, the PBS solution is a 0.2 mol/L, pH8.0 phosphatebuffer with a volume of 5-8 mL.

In one embodiment, the dialyzing and lyophilzing process is as follows:filling and sealing the complete antigen of aflatoxin biosyntheticprecursor ST in aqueous phase into a dialysis bag, and dialyzing against0.01-0.02 mol/L, pH8.0 phosphate buffer for a total of 3 days, where thedialysis solution is replaced once every 4-12 h; after completion of thelast dialysis, separating the solution in the dialysis bag intofractions, and lyophilizing the fractions, to obtain the artificialantigen of aflatoxin biosynthetic precursor ST.

In one embodiment, the carrier protein may be selected from biologicalproteins or synthetic polypeptides, such as bovine serum albumin, eggwhite albumin, hemocyanin, and polylysine.

In one embodiment, a method for preparing the artificial antigen ofaflatoxin biosynthetic precursor ST includes:

(1) weighing 0.1 g of commercially-available hydroxyacetic acid anddissolving it in 0.4 mL of trifluoroacetic acid; weighing 1 mg ofaflatoxin biosynthetic precursor ST and dissolving it in 0.4 mL ofacetonitrile to obtain a solution of aflatoxin biosynthetic precursor STin acetonitrile; drawing the solution of aflatoxin biosyntheticprecursor ST in acetonitrile with a syringe, pipetting gently thesolution into the mixture of hydroxyacetic acid/trifluoroacetic acid,reacting at room temperature for 4 h with stirring; evaporating thesolvent rotationally, to obtain a light yellowish green oily substancewhich is the aflatoxin biosynthetic precursor ST hapten;

(2) weighing 1 mg of aflatoxin biosynthetic precursor ST hapten and 4 mgof N-hydroxylsuccinimide, placing them in a reaction flask and reactingfor 1 h; weighing 7 mg of carbodiimide and dissolving it in 0.2 mL of1,4-dioxohextane to obtain a solution of carbodiimide in1,4-dioxohextane, then adding the solution dropwise into the reactionflask and reacting at room temperature for 4 h until a white precipitateis generated in the reaction flask; after completion of the reaction,allowing the reactants to stand at room temperature overnight, and inthe next day, centrifuging at 8000 r/min for 5 min and taking thesupernatant; weighing 4 mg of bovine serum albumin and dissolving it in5 mL 0.2 mol/L, pH8.0 PBS buffer, adding the above-mentioned supernatantdropwise into the PBS solution containing bovine serum albumin, afterthe sample addition is completed, running the reaction for 4 h to obtainan complete antigen of aflatoxin biosynthetic precursor ST in aqueousphase; and

(3) filling and enclosing the complete antigen of aflatoxin biosyntheticprecursor ST in aqueous phase from the above-mentioned step (2) into adialysis bag and dialyzing against 0.01 mol/L, pH8.0 PBS buffer for atotal of 3 days, the dialyzing solution is replaced once every 12 h;after completion of the last dialysis, the solution in the dialysis bagis separated to fractions into centrifuge tubes, and lyophilizing thefractions to obtain the artificial antigen of aflatoxin biosyntheticprecursor ST, i.e., aflatoxin biosynthetic precursor ST-bovine serumalbumin.

In one embodiment, using BSA as the carrier protein, the synthesis routeof coupling aflatoxin biosynthetic precursor ST and carrier protein toobtain the artificial antigen of aflatoxin biosynthetic precursor ST(aflatoxin biosynthetic precursor ST-BSA) is as follows:

Certain embodiments of the present invention, among other things, havethe following beneficial advantages.

1. A double-carbon-atoms arm structure (i.e., carboxymethoxy) isconnected to the ST molecule of the artificial antigen of aflatoxinbiosynthetic precursor ST synthesized in the present invention. Thisfacilitates sufficient exposure of the ST molecule on the carrierprotein, reduces the effect of the coupled carrier protein on theimmunogenicity of the artificial antigen, can have the mice immunizedmore efficiently to produce antibodies with high sensitivity and strongspecificity against aflatoxin biosynthetic precursor ST, and is ofimportant and practical significance in rapid detection of aflatoxinbiosynthetic precursor ST with immunological analysis techniques. 2. Thewhole preparation process in the present invention is simple andpracticable, does not need special instrumentation, has low cost, andthus is easy for scale up production and easy in widen applications. 3.The artificial antigen of aflatoxin biosynthetic precursor STsynthesized according to certain embodiments of the present invention ismore stable.

These and other aspects of the present invention will become apparentfrom the following description of the preferred embodiment taken inconjunction with the following drawings, although variations andmodifications therein may be effected without departing from the spiritand scope of the novel concepts of the disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings illustrate one or more embodiments of theinvention and together with the written description, serve to explainthe principles of the invention. Wherever possible, the same referencenumbers are used throughout the drawings to refer to the same or likeelements of an embodiment, and wherein:

FIG. 1 is an ultraviolet-visible spectrum continuous scanning profile inone embodiment of the present invention, in which ST is theultraviolet-visible spectrum continuous scanning profile of aflatoxinbiosynthetic precursor ST; BSA is the ultraviolet-visible spectrumcontinuous scanning profile of bovine serum albumin; and ST-BSA is theultraviolet-visible spectrum continuous scanning profile of theartificial antigen of aflatoxin biosynthetic precursor ST.

DETAINED DESCRIPTION OF THE INVENTION

The present invention is more particularly described in the followingexamples that are intended as illustrative only since numerousmodifications and variations therein will be apparent to those skilledin the art. Various embodiments of the invention are now described indetail. Referring to the drawings, like numbers indicate like componentsthroughout the views. As used in the description herein and throughoutthe claims that follow, the meaning of “a”, “an”, and “the” includesplural reference unless the context clearly dictates otherwise. Also, asused in the description herein and throughout the claims that follow,the meaning of “in” includes “in” and “on” unless the context clearlydictates otherwise. Moreover, titles or subtitles may be used in thespecification for the convenience of a reader, which shall have noinfluence on the scope of the present invention.

Furthermore, relative terms, such as “lower” or “bottom” and “upper” or“top,” may be used herein to describe one element's relationship toanother element as illustrated in the Figures. It will be understoodthat relative terms are intended to encompass different orientations ofthe device in addition to the orientation depicted in the Figures. Forexample, if the device in one of the figures is turned over, elementsdescribed as being on the “lower” side of other elements would then beoriented on “upper” sides of the other elements. The exemplary term“lower”, can therefore, encompasses both an orientation of “lower” and“upper,” depending of the particular orientation of the figure.Similarly, if the device in one of the figures is turned over, elementsdescribed as “below” or “beneath” other elements would then be oriented“above” the other elements. The exemplary terms “below” or “beneath”can, therefore, encompass both an orientation of above and below.

As used herein, “around”, “about”, “substantially” or “approximately”shall generally mean within 20 percent, preferably within 10 percent,and more preferably within 5 percent of a given value or range.Numerical quantities given herein are approximate, meaning that the term“around”, “about”, “substantially” or “approximately” can be inferred ifnot expressly stated.

As used herein, the terms “comprising”, “including”, “carrying”,“having”, “containing”, “involving”, and the like are to be understoodto be open-ended, i.e., to mean including but not limited to.

Example 1 An Artificial Antigen of Aflatoxin Biosynthetic Precursor STwas Obtained by the Following Steps

(1) 0.1 g of commercially-available hydroxyacetic acid (with watercontent of approximately 1%) was weighed and dissolved in 0.4 mL oftrifluoroacetic acid; 1 mg of aflatoxin biosynthetic precursor ST wasweighed and dissolved in 0.4 mL of acetonitrile; the solution ofaflatoxin biosynthetic precursor ST in acetonitrile was drawn with asyringe, and pipetted gently into the mixture of hydroxyaceticacid/trifluoroacetic acid and reacted at room temperature (20° C.-30°C.) for 4 h with magnetic stirring; the solvent was evaporatedrotationally, yielding a light yellowish green oily substance which wasthe aflatoxin biosynthetic precursor ST hapten.

(2) 1 mg of aflatoxin biosynthetic precursor ST hapten and 4 mg ofN-hydroxylsuccinimide were weighed and placed into a reaction flask, andreacted at room temperature for 1 h with magnetic stirring; 7 mg ofcarbodiimide was weighed and dissolved in 0.2 mL of 1,4-dioxohextane;the solution of carbodiimide in 1,4-dioxohextane was added slowlydropwise into the reaction flask and reacted at room temperature for 4 hwith magnetic stirring until a white precipitate was generated in thereaction flask; after completion of the reaction, the reactants werekept at room temperature overnight, in the next day, the reactants weresubjected to centrifugation at 8000 r/min for 5 min, and the supernatantwas obtained; 4 mg of bovine serum albumin (BSA) was weighed anddissolved in 5 mL of PBS buffer (0.2 mol/L, pH8.0), the above obtainedsupernatant was added dropwise into the solution of BSA in PBS; afterfinishing addition of the supernatant, the reaction was run for 4 h,yielding an complete antigen of aflatoxin biosynthetic precursor ST inaqueous phase.

(3) The complete antigen of aflatoxin biosynthetic precursor ST inaqueous phase obtained from the above-mentioned step (2) was sealed intoa dialysis bag and dialyzed for a total of 3 days against 0.01 mol/L,pH8.0 phosphate buffer, where the dialysis solution was replaced onceevery 12 h; after completion of the last dialysis, the solution in thedialysis bag was divided into fractions, placed in centrifuge tubes, andlyophilized to yield the artificial antigen of aflatoxin biosyntheticprecursor ST: aflatoxin biosynthetic precursor ST-bovine serum albumin.The ultraviolet-visible spectrum continuous scanning profile of theabove artificial antigen can be seen in FIG. 1.

Example 2 An Artificial Antigen of Aflatoxin Biosynthetic Precursor STwas Obtained by the Following Steps

(1) 0.5 g of commercially-available hydroxyacetic acid (with watercontent of approximately 1%) was weighed and dissolved in in 2.2 mL oftrifluoroacetic acid; 5 mg of aflatoxin biosynthetic precursor ST wasweighed and dissolved in 2.2 mL of acetonitrile; the solution ofaflatoxin biosynthetic precursor ST in acetonitrile was drawn with asyringe, and pipetted gently into the mixture of hydroxyaceticacid/trifluoroacetic acid and reacted at room temperature for 5 h withmagnetic stirring; the solvent was evaporated rotationally, yielding alight yellowish green oily substance which was the aflatoxinbiosynthetic precursor ST hapten.

(2) 5.3 mg of aflatoxin biosynthetic precursor ST hapten and 17 mg ofN-hydroxylsuccinimide were weighed and placed into a reaction flask, andreacted at room temperature for 1.5 h with magnetic stirring; 26 mg ofcarbodiimide was weighed and dissolved in 0.7 mL of 1,4-dioxohextane;the solution of carbodiimide in 1,4-dioxohextane was added slowlydropwise into the reaction flask and reacted at room temperature for 4.5h with magnetic stirring until a white precipitate was generated in thereaction flask; after completion of the reaction, the reactants werekept at room temperature overnight, in the next day, the reactants weresubjected to centrifugation at 8000 r/min for 5 min, and the supernatantwas obtained; 18 mg of ovalbumin (OVA) was weighed and dissolved in 6.5mL of PBS buffer (0.2 mol/L, pH8.0), the above-obtained supernatant wasadded dropwise into the solution of OVA in PBS; after finishing theaddition of the supernatant, the reaction was run for 4.5 h, yielding ancomplete antigen of aflatoxin biosynthetic precursor ST in aqueousphase.

(3) The complete antigen of aflatoxin biosynthetic precursor ST inaqueous phase obtained from the above-mentioned step (2) was sealed intoa dialysis bag and dialyzed for a total of 3 days against 0.015 mol/L,pH8.0 phosphate buffer, where the dialysis solution was replaced onceevery 8 h; after completion of the last dialysis, the solution in thedialysis bag was divided into fractions, placed in centrifuge tubes, andlyophilized to yield the artificial antigen of aflatoxin biosyntheticprecursor ST: aflatoxin biosynthetic precursor ST-OVA.

Example 3 An Artificial Antigen of Aflatoxin Biosynthetic Precursor STwas Obtained by the Following Steps

(1) 1 g of commercially-available hydroxyacetic acid (with water contentof approximately 1%) was weighed and dissolved in 4 mL oftrifluoroacetic acid; 10 mg of aflatoxin biosynthetic precursor ST wasweighed and dissolved in 4 mL of acetonitrile; the solution of aflatoxinbiosynthetic precursor ST in acetonitrile was drawn with a syringe, andpipetted gently into the mixture of hydroxyacetic acid/trifluoroaceticacid and reacted at room temperature for 6 h with magnetic stirring; thesolvent was evaporated rotationally, yielding a light yellowish greenoily substance which was the aflatoxin biosynthetic precursor ST hapten.

(2) 10 mg of aflatoxin biosynthetic precursor ST hapten and 30 mg ofN-hydroxylsuccinimide were weighed and placed into a reaction flask, andreacted at room temperature for 2 h with magnetic stirring; 45 mg ofcarbodiimide was weighed and dissolved in 1.2 mL of 1,4-dioxohextane;the solution of carbodiimide in 1,4-dioxohextane was added slowlydropwise into the reaction flask and reacted at room temperature for 5 hwith magnetic stirring until a white precipitate was generated in thereaction flask; after completion of the reaction, the reactants werekept at room temperature overnight, in the next day, the reactants weresubjected to centrifugation at 8000 r/min for 5 min, and the supernatantwas obtained; 30 mg of hemocyanin (KLH) was weighed and dissolved in 8mL of PBS buffer (0.2 mol/L, pH8.0), the above-mentioned supernatant wasadded dropwise into the solution of KLH in PBS; after finishing theaddition of the supernatant, the reaction was run for 5 h, yielding ancomplete antigen of aflatoxin biosynthetic precursor ST in aqueousphase.

(3) The complete antigen of aflatoxin biosynthetic precursor ST inaqueous phase obtained from the above-mentioned step (2) was sealed intoa dialysis bag and dialyzed for a total of 3 days against 0.02 mol/L,pH8.0 phosphate buffer, where the dialysis solution was replaced onceevery 4 h; after completion of the last dialysis, the solution in thedialysis bag was divided into fractions, placed into centrifuge tubes,and lyophilized to yield the artificial antigen of aflatoxinbiosynthetic precursor ST: aflatoxin biosynthetic precursor ST-KLH.

Identification of the artificial antigen according to certainembodiments of the present invention is as follows.

1. The artificial antigen of aflatoxin biosynthetic precursor ST wasidentified by the ultraviolet-visible spectrum continuous scanningprofile. As shown in FIG. 1, the artificial antigen of aflatoxinbiosynthetic precursor ST was coupled successfully to the carrierprotein, bovine serum albumin. From the absorbance value and theextinction coefficient at the characteristic ultraviolet absorptionwavelength of 413 nm for the conjugate, the coupling ratio between theaflatoxin biosynthetic precursor ST and the bovine serum albumin iscalculated to be 3.4:1.

2. Immunization in animal had confirmed that anti-aflatoxin biosyntheticprecursor ST antibodies were produced.

(1) Immunological experiment in mice: the above-mentioned synthesizedaflatoxin biosynthetic precursor ST-bovine serum albumin was formulatedwith 0.85% of physiological saline into a solution of 0.67 mg/mL. Thefirst immunization was performed by mixing 0.45 mL Freund's completeadjuvant with an equal volume of the formulated aflatoxin biosyntheticprecursor ST-bovine serum albumin mentioned above and fully emulsifyingthe mixture, followed by subcutaneous injection of 0.3 mL of thepreparation (equivalent to 100 μg protein) into each of the 6-8weeks-old Balb/c mice. Immunization was boosted 1 time every 3 weeks andin the booster immunization, the adjuvant would be changed to Freund'sincomplete adjuvant with the remaining of the procedure being the sameas those in the first immunization method. Blood was drawn from the tailof the mouse 7-10 days after each booster immunization and the antiserawere prepared.

(2) Determination of the antibody titer by non-competitive ELISA assay:the coated antigen, aflatoxin biosynthetic precursor ST-bovine serumalbumin, was diluted with a pH9.6 carbonate salt buffer to 0.5 μg/mL.100 μl of the diluted solution was added into each of the wells of theELISA plate. The plate was incubated at 4° C. overnight, and then thecoating solution was poured off. Each well was washed three times with atypical phosphate-Tween washing liquid and dripped to dry. 200 μl of1.5% skimmed milk solution was added into each well and the plate wasblocked at 37° C. for 2 h. The blocking solution was poured off, andeach well was washed three times and dripped to dry. The antiserum wassubjected to double dilution starting from 500 folds, and each well wasadded with 100 μl. The control wells were set up in parallel withnegative serum as the negative control and 0.15 mol/L, pH7.4 phosphatebuffer as the blank control. The plate was incubated and moisturized at37° C. for 2 h, washed three times, and dripped to dry. 100 μl of theenzymatically labelled goat-anti-mouse secondary antibody IgG:HRPdiluted at 1:5000 with 0.15 mol/L, pH7.4 phosphate buffer was added intoeach well, and incubated and moisturized at 37° C. for 2 h. Each wellwas washed six times and dripped to dry. 100 μl of the reactivesubstrate solution was added into each well and reacted at 37° C. in thedark for 10-15 min. Then, 50 μl of the 2 mol/L sulphuric acid solutionwas added into each well to stop the reaction. After 5 min, zero is setusing the blank control well, and the absorbance value was measured at450 nm. The antiserum titer was the dilution factor of the antiserumcorresponding to the measured absorbance value of the antiserum twotimes that of the negative serum. The results were listed in Table 1.

TABLE 1 The antiserum titer against aflatoxin biosynthetic precursor STNeg- Dilution fold ative 1250 2500 5000 10000 20000 40000 80000 160000control Ab- 1.16 1.14 1.07 0.85 0.62 0.43 0.39 0.14 0.09 sorb- ance

From the data in Table 1, it can be proved that the artificial antigenof aflatoxin biosynthetic precursor ST prepared according to the methodof the invention can produce an antiserum with a titer of greater than80000 after immunization.

(3) ELISA competitive inhibition assay: the operation steps in the ELISAassay were the same as above, except that the antisera subjected todouble dilution were replaced with the antiserum solutions containingdifferent concentrations of the standard of the aflatoxin biosyntheticprecursor ST. It would confirm that the antibodies in the antisera wascapable of binding to the aflatoxin biosynthetic precursor ST if theabsorbance value was decreased with increase in the concentrations ofthe standard of the aflatoxin biosynthetic precursor ST. Resultsobtained from the competitive ELISA assay were shown in the followingTable 2:

TABLE 2 ELISA competitive inhibition assay results against aflatoxinbiosynthetic precursor ST Inhibition concentrations for aflatoxinbiosynthetic precursor ST (ng/mL) 0 0.01 0.04 0.14 0.41 1.23 3.70 11.133.3 100 Ab- 0.90 0.82 0.75 0.66 0.49 0.34 0.11 0.03 0.01 0 sorb- ance

Results of competitive ELISA inhibition assay in Table 2 indicates thatthe antibody against aflatoxin biosynthetic precursor ST had beenproduced in the mice, thereby demonstrating that the artificial antigenof aflatoxin biosynthetic precursor ST prepared according to the methodin the present invention is successful. Further, it can be seen fromTable 2 that IC₅₀ value of the antibody for the aflatoxin biosyntheticprecursor ST is 0.41 ng/mL. It demonstrates that antibodies withhigh-sensitivity can be produced after immunization of the mice with theartificial antigen of aflatoxin biosynthetic precursor ST synthesizedaccording to the method in the present invention.

(4) Antibody specificity assay: the operation steps of ELISA were thesame as above, except that the antisera subjected to double dilutionwere replaced with the antiserum solutions containing differentconcentrations of the standards of aflatoxins B1, B2, G1, and G2. Itwould confirm that the antibodies in the antisera did not bind toaflatoxins B1, B2, G1, and G2 if there were no regular changes in theabsorbance values with the increase in the concentrations of thestandards of aflatoxins B1, B2, G1, and G2. Results obtained from thecompetitive ELISA assay were shown in the following Table 3.

TABLE 3 ELISA competitive inhibition assay against aflatoxins B1, B2,G1, and G2 Inhibition concentrations for aflatoxin B1 (ng/mL) 0 0.010.04 0.14 0.41 1.23 3.70 11.1 33.3 100 Absorbance 0.99 1.01 0.95 0.931.05 0.97 0.92 0.96 0.99 0.94 Inhibition concentrations for aflatoxin B2(ng/mL) 0 0.01 0.04 0.14 0.41 1.23 3.70 11.1 33.3 100 Absorbance 1.041.06 0.99 1.07 1.01 1.02 1.04 1.01 1.02 1.08 Inhibition concentrationsfor aflatoxin G1 (ng/mL) 0 0.01 0.04 0.14 0.41 1.23 3.70 11.1 33.3 100Absorbance 1.02 1.07 1.05 1.01 0.98 1.04 1.09 1.11 1.05 1.07 Inhibitionconcentrations for aflatoxin G2 (ng/mL) 0 0.01 0.04 0.14 0.41 1.23 3.7011.1 33.3 100 Absorbance 1.08 1.04 1.05 1.04 1.01 1.09 1.02 1.10 1.011.03

Results of the experiment on the specificity of the antibody in Table 3indicated that the antibody produced in the mice did not bind toaflatoxins B1, B2, G1, and G2, which demonstrated that antibodies withhigh specificity can be produced after immunization of the mice with theartificial antigen of aflatoxin biosynthetic precursor ST synthesizedaccording to the method in the present invention.

The foregoing description of the exemplary embodiments of the inventionhas been presented only for the purposes of illustration and descriptionand is not intended to be exhaustive or to limit the invention to theprecise forms disclosed. Many modifications and variations are possiblein light of the above teaching.

The embodiments are chosen and described in order to explain theprinciples of the invention and their practical application so as toactivate others skilled in the art to utilize the invention and variousembodiments and with various modifications as are suited to theparticular use contemplated. Alternative embodiments will becomeapparent to those skilled in the art to which the present inventionpertains without departing from its spirit and scope. Accordingly, thescope of the present invention is defined by the appended claims ratherthan the foregoing description and the exemplary embodiments describedtherein.

What is claimed is:
 1. An artificial antigen of aflatoxin biosyntheticprecursor sterigmatocystin (ST), obtained by coupling an aflatoxinbiosynthetic precursor ST to a carrier protein, wherein the step ofcoupling comprises: opening a double bound of the difuran ring of theaflatoxin biosynthetic precursor ST; connecting an active carboxymethoxygroup to the opened double bond of the difuran ring; and connecting thecarboxyl on the active carboxymethoxy group to an amino group on thecarrier protein.
 2. A method for preparing the artificial antigen ofaflatoxin biosynthetic precursor ST of claim 1, comprising: reactinghydroxyacetic acid with the double bound of the difuran ring in theaflatoxin biosynthetic precursor ST to obtain an aflatoxin biosyntheticprecursor ST hapten with the active carboxymethoxy group; attaching thecarboxyl group on the aflatoxin biosynthetic precursor ST hapten to theamino group on the carrier protein at room temperature via an activeester procedure to form an attached mixture; and dialyzing andlyophilizing the attached mixture to obtain the artificial antigen ofaflatoxin biosynthetic precursor ST.
 3. The method for preparing theartificial antigen of aflatoxin biosynthetic precursor ST of claim 2,wherein the step of reacting the hydroxyacetic acid with the doublebound of the difuran ring in the aflatoxin biosynthetic precursor STcomprises: weighing 0.1-1 g of the hydroxyacetic acid and dissolving theweighed hydroxyacetic acid in 0.4-4 mL of trifluoroacetic acid to obtaina mixture of hydroxyacetic acid/trifluoroacetic acid; weighing 1-10 mgof the aflatoxin biosynthetic precursor ST and dissolving the weighedaflatoxin biosynthetic precursor ST in 0.4-4 mL of acetonitrile toobtain a solution of aflatoxin biosynthetic precursor ST inacetonitrile; drawing the solution of aflatoxin biosynthetic precursorST in acetonitrile with a syringe and pipetting gently into the mixtureof hydroxyacetic acid/trifluoroacetic acid, and allowing a reaction torun at room temperature for 4-6 hours under stirring; and aftercompletion of the reaction, evaporating rotationally the solvent toobtain a light yellowish green oily substance which is the aflatoxinbiosynthetic precursor ST hapten.
 4. The method for preparing theartificial antigen of aflatoxin biosynthetic precursor ST of claim 2,wherein the step of attaching the carboxyl group on the aflatoxinbiosynthetic precursor ST hapten to the amino group on the carrierprotein comprises: weighing 1-10 mg of the aflatoxin biosyntheticprecursor ST hapten and 4-30 mg of N-hydroxylsuccinimide, placing into areaction flask, and reacting for 1-2 h; weighing 7-45 mg of carbodiimideand dissolving in 0.2-1.2 mL of 1,4-dioxohextane to obtain a solution ofcarbodiimide in 1,4-dioxohextane, adding the solution of carbodiimide in1,4-dioxohextane dropwise into the reaction flask, reacting at roomtemperature for 4-5 hours until a white precipitate is generated in thereaction flask, after completion of the reaction, keeping reactants inthe reaction flask at room temperature overnight, and on the next day,centrifuging the reactants in the reaction flask to obtain asupernatant; and adding the supernatant dropwise into a phosphatebuffered saline (PBS) solution having 4-30 mg of the carrier proteindissolved therein, after the supernatant is added completely, allowingthe reaction to run for 4-5 hours to obtain an complete antigen ofaflatoxin biosynthetic precursor ST in aqueous phase.
 5. The method forpreparing the artificial antigen of aflatoxin biosynthetic precursor STof claim 4, wherein the PBS solution is 5-8 mL of a 0.2 mol/L, pH8.0phosphate buffer.
 6. The method for preparing the artificial antigen ofaflatoxin biosynthetic precursor ST of claim 2, wherein the step ofdialyzing and lyophilizing comprises: sealing the complete antigen ofaflatoxin biosynthetic precursor ST in aqueous phase into a dialysis bagand dialyzing for a total of 3 days against a dialysis solution of0.01-0.02 mol/L, pH8.0 phosphate buffer, wherein the dialysis solutionis replaced once every 4-12 hours; and after completion of the lastdialysis, dividing the solution in the dialysis bag into fractions, andlyophilizing the fractions to obtain the artificial antigen of aflatoxinbiosynthetic precursor ST.
 7. The method for preparing the artificialantigen of aflatoxin biosynthetic precursor ST of claim 2, comprising:(1) weighing 0.1 g of commercially-available hydroxyacetic acid anddissolvinged in 0.4 mL of trifluoroacetic acid to obtain a mixture ofhydroxyacetic acid/trifluoroacetic acid; weighing 1 mg of aflatoxinbiosynthetic precursor ST and dissolving in 0.4 mL of acetonitrile toobtain a solution of aflatoxin biosynthetic precursor ST inacetonitrile; drawing the solution of aflatoxin biosynthetic precursorST in acetonitrile with a syringe, pipetted gently into the mixture ofhydroxyacetic acid/trifluoroacetic acid and allowing a reaction to runat room temperature for 4 hours under stirring; evaporated rotationallythe solvent to obtain a light yellowish green oily substance which isthe aflatoxin biosynthetic precursor ST hapten; (2) weighing 1 mg of theaflatoxin biosynthetic precursor ST hapten and 4 mg ofN-hydroxylsuccinimide, placing into a reaction flask, and reacting for 1hour; weighing 7 mg of carbodiimide and dissolving in 0.2 mL of1,4-dioxohextane to obtain a solution of carbodiimide in1,4-dioxohextane, adding the solution of carbodiimide in1,4-dioxohextane dropwise into the reaction flask, reacting at roomtemperature for 4 hours until a white precipitate is generated in thereaction flask; after completion of the reaction, keeping reactants inthe reaction flask at room temperature overnight, and on the next daycentrifuging the reactants at 8000 r/min for 5 min, to obtain asupernatant; weighing 4 mg of bovine serum albumin and dissolving in 5mL of 0.2 mol/L, pH8.0 phosphate buffer, adding the supernatant dropwiseinto the phosphate buffer containing the bovine serum albumin, after thesupernatant is added completely, allowing the reaction to run for 4hours to obtain an complete antigen of aflatoxin biosynthetic precursorST in aqueous phase; and (3) sealing the complete antigen of aflatoxinbiosynthetic precursor ST in aqueous phase from the above step (2) intoa dialysis bag and dialyzing for a total of 3 days against a dialysissolution of 0.01 mol/L, pH8.0 phosphate buffer, wherein the dialysissolution is replaced once every 12 hours; after completion of the lastdialysis, dividing the solution in the dialysis bag into centrifugetubes, and lyophilizing to obtain the artificial antigen of aflatoxinbiosynthetic precursor ST, that is, aflatoxin biosynthetic precursorST-bovine serum albumin.